Mitochondrial diseases are devastating disorders for which there is no cure and no proven treatment. About 1 in 2000 individuals are at risk of developing a mitochondrial disease sometime in their lifetime. Half of those affected are children who show symptoms before age 5 and approximately 80% of these will die before age 20. The human suffering imposed by mitochondrial and metabolic diseases is enormous, yet much work is needed to understand the genetic and environmental causes of these diseases. Mitochondrial genetic diseases are characterized by alterations in the mitochondrial genome, as point mutations, deletions, rearrangements, or depletion of the mitochondrial DNA (mtDNA). The mutation rate of the mitochondrial genome is 10-20 times greater than of nuclear DNA, and mtDNA is more prone to oxidative damage than is nuclear DNA. Mutations in human mtDNA cause premature aging, severe neuromuscular pathologies and maternally inherited metabolic diseases, and influence apoptosis. The primary goal of this project is to understand the contribution of the replication apparatus in the production and prevention of mutations in mtDNA. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we have focused this project on understanding the role of the human pol gamma in mtDNA mutagenesis. Human mitochondrial DNA is replicated by the two-subunit gamma, composed of a 140 kDa subunit containing catalytic activity and a 55 kDa accessory subunit. The catalytic subunit contains DNA polymerase activity, 3'-5' exonuclease proofreading activity, and 5'dRP lyase activity required for base excision repair. As the only DNA polymerase in animal cell mitochondria, pol gamma participates in DNA replication and DNA repair. The 140 kDa catalytic subunit for pol gamma is encoded by the nuclear POLG gene. To date nearly 250 pathogenic mutations in POLG that cause a wide spectrum of disease including Progressive external ophthalmoplegia (PEO), parkinsonism, premature menopause, Alpers syndrome, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) or sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO). Heterozygous mutations in POLG2 are responsible for several mitochondrial diseases, including progressive external ophthalmoplegia (PEO). We have previously analyzed four such POLG2 mutations with homodimeric preparations of purified recombinant p55 variants and documented their biochemical defects in vitro. However, because the affinity between monomers within the p55 dimer is extremely high (estimated intermolecular Kd of < 0.1 nM) and these mutations occur in heterozygous states, affected individuals would harbor mixtures of variant and wild-type molecules. We expect p55 to occur as 50% heterodimers, 25% wild-type homodimers, and 25% variant homodimers. To analyze the consequences of heterodimeric variants in vitro, we constructed a novel vector with one gene coding for p55 with an N-terminal Strep-tag and a second p55 gene encoding an N-terminal hexa histidine-tag. We also developed a method to purify heterodimeric p55 proteins harboring the two tags. Processivity and primer extension experiments revealed that WT-p55/p55-G451E heterodimers fail to stimulate p140. These experiments support our hypothesis that in vivo dysfunctions result from dominant negative proteins that negatively affect or block the function of wild-type gene products. To complement the biochemical studies and to address the consequences of these mutations in vivo, we also developed a novel HEK293 cell model to monitor p55 expression and localization using green fluorescent protein (GFP) fused to various p55 variants. As a negative control for targeting we removed the N-terminal mitochondrial targeting sequence. The GFP-p55 molecules lacking mitochondrial targeting sequences have diffuse fluorescence throughout the cytoplasm indicating a failure to localize within mitochondria. Interestingly, two clinically severe variants, L475DfsX2 and P205R p55, were able to enter mitochondria but had diffuse fluorescence within the mitochondrial network. Unlike wild-type p55 molecules, which colocalize with DAPI-stained mtDNA as punctate nucleoid structures located within the mitochondrial matrix, the diffuse fluorescence of the two severe variants indicates a failure to associate with mtDNA. The stable cell lines developed here will allow characterization of the mechanisms of these complex diseases by monitoring mtDNA maintenance and cellular physiology. Additionally, the ongoing biochemical studies analyzing p55 variant heterodimers will clarify other possible dominant negative interactions between Polg disease subunits. This research will improve our knowledge of how p55 variants contribute to mtDNA instability and PEO. On a broader scale these techniques could be applied to understanding mitochondrial dysfunction associated with aging, neurodegenerative diseases and cancers. Mitochondrial DNA (mtDNA) encodes proteins essential for ATP production. Mutant variants of the mtDNA polymerase cause mutagenesis that contributes to aging, genetic diseases, and sensitivity to environmental agents. We interrogated mtDNA replication in Saccharomyces cerevisiae strains with disease-associated mutations affecting conserved regions of the mtDNA polymerase, Mip1. Mutant frequency arising from mitochondrial base substitutions that confer erythromycin resistance and deletions between 21-nucleotide direct repeats was determined. Previously, increased mutagenesis was observed in strains encoding mutant variants that were insufficient to maintain mtDNA and that were not expected to reduce polymerase fidelity or exonuclease proofreading. Increased mutagenesis could be explained by mutant variants stalling the replication fork, thereby predisposing the template DNA to irreparable damage that is bypassed with poor fidelity. This hypothesis suggests that the exogenous base-alkylating agent, methyl methanesulfonate (MMS), would further increase mtDNA mutagenesis. Mitochondrial mutagenesis associated with MMS exposure was increased up to 30-fold in mip1 mutants containing disease-associated alterations that affect polymerase activity. Disrupting exonuclease activity of mutant variants was not associated with increased spontaneous mutagenesis compared with exonuclease-proficient alleles, suggesting that most or all of the mtDNA was replicated by wild type Mip1. A novel subset of C to G transversions was responsible for about half of the mutants arising after MMS exposure implicating error-prone bypass of methylated cytosines as the predominant mutational mechanism. Exposure to MMS does not disrupt exonuclease activity that suppresses deletions between 21-nucleotide direct repeats, suggesting the MMS-induce mutagenesis is not explained by inactivated exonuclease activity. Further, trace amounts of CdCl2 inhibit mtDNA replication but suppresses MMS-induced mutagenesis. These results suggest a novel mechanism wherein mutations that lead to hypermutation by DNA base-damaging agents and associate with mitochondrial disease may contribute to previously unexplained phenomena, such as the wide variation of age of disease onset and acquired mitochondrial toxicities.